David C. Rayner

Epithelial to Mesenchymal Transition During Late Deterioration of Human Kidney Transplants: The Role of Tubular Cells in Fibrogenesis

The hallmark of failing renal transplants is tubular atrophy and interstitial fibrosis (TA/IF). Injury to tubular epithelial cells (TEC) could contribute to fibrogenesis via epithelial–mesenchymal transition (EMT). We examined the features of EMT in renal transplants that developed TA/IF. Biopsies from 10 allograft kidneys with impaired function and TA/IF and 10 biopsies from transplants with stable function were compared to their implantation biopsies. Relative to implantation biopsies, TEC in TA/IF kidneys showed loss of epithelial markers (E‐cadherin, cytokeratin) with altered distribution. Some TEC also showed new cytoplasmic expression of mesenchymal markers vimentin, S100A4, and alpha smooth muscle actin (α‐SMA) and collagen synthesis marker heat shock protein (HSP‐47), both in deteriorating and atrophic tubules. Double immunostaining showed coexpression of cytokeratin and vimentin, S100A4 and HSP‐47, indicating intermediate stages of EMT in TA/IF. These changes were absent or much less in transplants with stable function. EMT features in the TA/IF group correlated with serum creatinine (vimentin, S100A4, HSP‐47), history of T‐cell‐mediated rejection (cytokeratin, S100A4) and proteinuria (cytokeratin). These findings support a model in which the TEC damage induces loss of epithelial features and expression of fibroblast features, as a common pathway of deterioration by either immunologic or nonimmunologic processes.

Glutaraldehyde-Fixed Bioprosthetic Heart Valve Conduits Calcify and Fail From Xenograft Rejection

Background— Glutaraldehyde fixation (G-F) decreases but likely does not eliminate the antigenicity of bioprosthetic heart valves. Rejection (with secondary dystrophic calcification) may be why G-F xenograft valves fail, especially in young patients, who are more immunocompetent than the elderly. Therefore, we sought to determine whether rejection of G-F xenograft occurs and to correlate this with graft calcification. Methods and Results— Ascending aortas/valves (from rats [syngeneic] or guinea pigs [xenogeneic]) were transplanted (fresh or after 48 hour of G-F) into the infrarenal aortas of young rat recipients for 20 days. A xenogeneic group was also treated with steroids until graft harvest. The valves and media/adventitia were scored blindly for inflammation (0 to 4). Percent graft infiltration by T cells/macrophages was determined (immunohistochemistry), and rat IgG ELISAs were performed. There was >3 times more valve inflammation, >10 times more valve T-cell/macrophage infiltrate, and >3 times antibody rise in the G-F xenogeneic groups compared with the fresh syngeneic or the G-F syngeneic groups (P<0.05). There was >2 times more adventitial inflammation and T-cell/macrophage infiltrate in the xenogeneic groups (P<0.05). Steroid treatment decreased inflammation and antibody rise in the xenogeneic groups (P<0.05). Correlation analysis revealed media/adventitia inflammation (P=0.02) and percent macrophage (P=0.01) infiltration to be predictors of calcification. Conclusions— G-F xenografts have cellular/humoral rejection and calcify secondarily.

Effects of Donor Age and Cell Senescence on Kidney Allograft Survival

The biological processes responsible for somatic cell senescence contribute to organ aging and progression of chronic diseases, and this may contribute to kidney transplant outcomes. We examined the effect of pre‐existing donor aging on the performance of kidney transplants, comparing mouse kidney isografts and allografts from old versus young donors. Before transplantation, old kidneys were histologically normal, but displayed an increased expression of senescence marker p16INK4a. Old allografts at day 7 showed a more rapid emergence of epithelial changes and a further increase in the expression of p16INK4a. Similar but much milder changes occurred in old isografts. These changes were absent in young allografts at day 7, but emerged by day 21. The expression of p16INK4a remained low in young kidney allografts at day 7, but increased with severe rejection at day 21. Isografts from young donors showed no epithelial changes and no increase in p16INK4a. The measurements of the alloimmune response—infiltrate, cytology, expression of perforin, granzyme B, IFN‐γ and MHC—were not increased in old allografts. Thus, old donor kidneys display abnormal parenchymal susceptibility to transplant stresses and enhanced induction of senescence marker p16INK4a, but were not more immunogenic. These data are compatible with a key role of somatic cell senescence mechanisms in kidney transplant outcomes by contributing to donor aging, being accelerated by transplant stresses, and imposing limits on the capacity of the tissue to proliferate.

Cardiac function, myocardial glutathione, and matrix metalloproteinase-2 levels in hypoxic newborn pigs reoxygenated by 21%, 50%, or 100% oxygen.

After asphyxia, it is standard to resuscitate the newborn with 100% oxygen, which may create a hypoxia-reoxygenation process that may contribute to subsequent myocardial dysfunction. We examined the effects of graded reoxygenation on cardiac function, myocardial glutathione levels, and matrix metalloproteinase (MMP)-2 activity during recovery. Thirty-two piglets (1-3 days old, weighing 1.5-2.1 kg) were anesthetized and instrumented for continuous monitoring of cardiac index, and systemic and pulmonary arterial pressures. After 2 h of hypoxia, piglets were randomized to receive reoxygenation for 1 h with 21%, 50%, or 100% oxygen (n = 8 each), followed by 3 h at 21% oxygen. At 2 h of hypoxemia (PaO2 32-34 mmHg), the animals had hypotension, decreased cardiac index, and elevated pulmonary arterial pressure (P < 0.001 vs. controls). Upon reoxygenation, cardiac function recovered in all groups with higher cardiac index and lower systemic vascular resistance in the 21% group at 30 min of reoxygenation (P < 0.05 vs. controls). Pulmonary artery pressure normalized in an oxygen-dependent fashion (100% = 50% > 21%), despite an immediate recovery of pulmonary vascular resistance in all groups. The hypoxia-reoxygenated (21%-100%) hearts had similarly increased MMP-2 activity and decreased glutathione levels (P < 0.05, 100% vs. controls), which correlated significantly with cardiac index and stroke volume during reoxygenation, and similar features of early myocardial necrosis. In neonatal resuscitation, if used with caution because of a slower resolution of pulmonary hypertension, 21% reoxygenation results in similar cardiac function and early myocardial injury as 50% or 100%. The significance of higher oxidative stress with high oxygen concentration is unknown, at least in the acute recovery period.

Light dosimetry using the P3 approximation

In earlier work, we demonstrated that radiance, calculated using the P3 approximation in a plane wave geometry, could be used to accurately predict the optical parameters of an Intralipid/methylene blue phantom. Plane wave geometry is impractical for clinical use but the results of this work encouraged us to further develop the P3 approximation for a spherical geometry, described in this paper. Radiance predicted by this model for a defined Intralipid/methylene blue phantom was compared with radiance measured in this phantom. The results demonstrate that the spherical derivation of the P3 approximation will reproducibly predict optical parameters of a tissue phantom as effectively as the slab geometry derivation of the P3 approximation. In a similar protocol, the P3 approximation was used to estimate the optical parameters of ex vivo human prostate. Radiance in this case was measured in the prostate samples using an after loading technique. Three prostate samples tested were found to be surprisingly optically homogeneous. The after loading protocol described in this paper could form the basis of a minimally invasive and effective clinical method to optically characterize human prostate.

BTLA targeting modulates lymphocyte phenotype, function, and numbers and attenuates disease in nonobese diabetic mice

The novel coinhibitory receptor BTLA may have a regulatory role in maintaining peripheral tolerance; however, its role in autoimmune diabetes is unknown. In this study, we show that anti‐BTLA mAb 6F7 selectively depleted pathogenic B and CD4+ TH cells; enhanced the proportion of cells with the forkhead box p3+ PD‐1+CD4+ regulatory T phenotype; and increased the production of potentially protective (IL‐10) and detrimental (IL‐2, IFN‐γ) cytokines in NOD mice. As interactions between BTLA and PD‐1 coinhibitory pathways have been described in the cardiac allograft model, we also investigated if these pathways may have significant interaction in autoimmune diabetes. Anti‐BTLA inhibited anti‐PD‐1‐potentiated total IL‐12 (p40+p70) production, suggesting the possibility that anti‐BTLA may have a greater effect in the setting of anti‐PD‐1‐triggered diabetes. To test this, NOD mice at 4 and 10 weeks of age were treated with anti‐BTLA mAb, anti‐PD‐1 mAb, both mAb, or isotype control and were monitored for diabetes development. Although anti‐BTLA mAb delayed diabetes onset significantly in 10‐ but not 4‐week‐old NOD mice, anti‐BTLA mAb attenuated anti‐PD‐1‐induced diabetes in both age groups. Hence, strategies targeting BTLA+ lymphocytes or therapies enhancing the BTLA‐negative cosignal may prove valuable in treating autoimmune diabetes.

Leiomyosarcoma of the bladder in a retinoblastoma patient.

We report a rare case of leiomyosarcoma in the bladder which occurred in an 18-year-old female with a prior history of retinoblastoma (RB). Molecular characterization of this tumor displayed a homozygous RB deletion and a reduced p53 expression. These results suggest that the loss of RB and p53 may have contributed to the initiation and/or progression of the leiomyosarcoma of the bladder in this patient.

Phosphoinositide 3-kinase β mediates microvascular endothelial repair of thrombotic microangiopathy

Thrombotic microangiopathy (TMA) commonly involves injury of kidney glomerular endothelial cells (ECs) and fibrin occlusion of the capillaries. The mechanisms underlying repair of the microvasculature and recovery of kidney function are poorly defined. In the developing vasculature, the phosphoinositide 3-kinase (PI3K) α isoform integrates many growth factor cues. However, the role of individual isoforms in repair of the established vasculature is unclear. We found that postnatal endothelial deletion of PI3Kβ sensitizes mice to lethal acute kidney failure after TMA injury. In vitro, PI3Kβ-deficient ECs show reduced angiogenic invasion of fibrin matrix with unaltered sensitivity to proapoptotic stress compared with wild-type ECs. This correlates with decreased expression of the EC tip cell markers apelin and Dll4 and is associated with a reduction in migration and proliferation. In vivo, PI3Kβ-knockdown ECs are deficient in assembly of microvessel-like structures. These data identify a critical role for endothelial PI3Kβ in microvascular repair following injury.

Noncytolytic human lymphocytes injure dermal microvessels in the huPBL-SCID skin graft model.

Recent transplantation experiments using perforin-deficient mice as allograft recipients have challenged the concept that allograft rejection is mediated exclusively by CTL. We sought to determine if human noncytolytic lymphocytes could mediate rejection of allogeneic human skin grafts in the huPBL-SCID mouse model of rejection. We generated short term lines of human lymphocytes from peripheral blood mononuclear cells using PHA as a mitogen. The first group was stimulated with PHA alone, the second with PHA plus IL-4 and neutralizing antibody to IL-12, and in the third group PBL were depleted of B cells and monocytes before stimulation as in group 2. After two passages, lines were tested for cytolytic ability and IFN-gamma production. Each line was injected i.p. to mice bearing allogeneic skin grafts. The grafts were harvested between day 16 and 21 after PBL injection, then the histology was scored by a blinded observer for degree of infiltration, microvessel injury, induction of epidermal MHC class II, and perforin expression. In vitro we found that PBL in groups 2 and 3 were unable to lyse cultured endothelial cells in a lectin-directed 111In release assay. In vivo 80% of the IL-4/anti-IL-12 groups maintained the IFN-gamma-low phenotype, and no perforin was detected in these grafts. Nevertheless, human microvessel injury was similar between the two groups. This was not antibody-dependent since the B-cell-depleted group showed similar injury. Moreover adjacent murine vessels were intact. We interpret these observations to show (1) these human PBL lines maintained their phenotype following in vivo restimulation, and (2) noncytolytic graft-infiltrating lymphocytes specifically promote injury of allogeneic human microvessels.

A comparison of combination dopamine and epinephrine treatment with high-dose dopamine alone in asphyxiated newborn piglets after resuscitation

Background:When asphyxiated neonates require additional cardiovascular support to moderate doses of dopamine infusion, controversy exists on the differential hemodynamic effects of two approaches (adding a second inotrope vs. increasing dopamine dosage). We hypothesized that high-dose dopamine (HD) would be detrimental to systemic and regional perfusion as compared with dopamine and epinephrine (D + E) combination therapy using a swine model of neonatal hypoxia–reoxygenation (H-R).Methods:Twenty-seven piglets (1–4 d, 1.5–2.5 kg) were used for continuous monitoring of systemic arterial pressure (mean arterial pressure, MAP) and pulmonary arterial pressure (PAP), cardiac output (cardiac index, CI), and carotid (carotid artery flow index, CAFI), superior mesenteric (superior mesenteric artery flow index), and renal arterial flows. H-R piglets underwent 2 h of hypoxia followed by 2 h of reoxygenation before drug infusion (2 h).Results:The hemodynamics of H-R piglets deteriorated gradually after reoxygenation. HD and D + E infusions improved CI similarly (both groups vs. control; P < 0.05). Both regimens increased MAP (P < 0.05) but not PAP, with decreased PAP/MAP ratio in D + E piglets. Both regimens improved CAFI and superior mesenteric artery flow index, with decreased mesenteric vascular resistance in HD-treated piglets. No significant effect on renal perfusion was observed.Conclusion:In H-R newborn piglets treated with a moderate dose of dopamine, adding epinephrine or further increasing dopamine improved systemic hemodynamics similarly; these treatments have differential effects on the pulmonary and mesenteric circulations.